Spectroscopy
Spectroscopy is the study of the absorption and emission of light. When physicists and chemists use the word spectroscopy, they are typically referring to infrared or wavelength-specific measurements of radiation. One specific type of spectroscopy is called "mass spectrometry" which is used in chemistry laboratories to identify the elemental composition and measure relative abundances of molecules following separation.
Just as a prism can be used to split white light into a rainbow, an instrument called a spectrometer can be used to break up electromagnetic radiation into its component wavelengths (or colors) like those in the visible spectrum. An instrument that breaks up light waves based on their frequencies rather than colors is called a "spectroscope. "This difference between instruments and spectroscopes is also reflected in the nomenclature of the instruments.
The electromagnetic spectrum is the range of frequencies that are measurable by instruments such as spectrophotometers, photometers or any other instrument with a light source, detector and a means to display frequency measurements. Each instrument's measurement scale depends on its wavelength range, accuracy requirements, and applicability. Spectroscopy is not limited to just these visible wavelengths because human vision covers the entire electromagnetic spectrum (from infrared to ultraviolet). The exact limits vary depending on many factors but are typically defined as being between 0.1 µm and 100 m (0.0025 inch to 3.28 ft), by far the visible part of the spectrum, and 0.5 nm to 100 nm (0.001 in to 3.28 ft), by far the infrared part of the spectrum.
All spectroscopy methods rely on absorbing energy from light sources and then converting it into a form that can be measured, either as an electrical voltage or as a specific absorption or emission of energy at some known wavelength which can be identified later on. This initial absorption leads to an electronic transition between two or more electronic states in which electrons drop down from higher-energy levels to lower-energy levels (a kind of thermal agitation) that is proportional to the wavelength of light being absorbed. The difference in energies between these states can be calculated, and the wavelengths at which they absorb or emit light can be plotted as a function of frequency. This spectrum can then be analyzed to identify the individual elements present in the sample and to determine relative abundances. The light source is usually monochromatic, although more complex sources exist that provide measuring capability over larger regions of the electromagnetic spectrum. Light sources are often collimated (which means they create narrow beams), so that only light from a specific direction or part of the sample is analyzed at once.
Spectroscope software interfaces provide an electronic system for designing measurement systems and for controlling these measurements. For example, the signal-versus-noise (S/N), signal-to-noise (S/N) ratio, and signal-to-noise with a reference video are used to describe the quality of a detector's signal. A high S/N is generally preferred to improve resolution, but a high S/N with a reference video may be more useful for identifying the cause of an incorrect measurement. Spectrometer models use various methods to calculate spectra. For example, modern models always provide at least two distinct channels of data, one for each unique wavelength on an instrument's measurement scale. In addition, some spectrometer models use a continuous wavelength representation rather than discrete wavelengths. Some spectral data can be displayed as intensity versus wavelength, which is a variation of the same data set.
Other detector models provide the ability to measure certain characteristics such as relative intensity and area under the curve (AUC). Area under the curve can be measured accurately only if an instrument's detector has an internal and stable video reference signal. Some spectroscope software interfaces also provide image control functions that allow users to minimize system installation costs and maximize measurements by eliminating expensive calibration equipment from their system designs. This process of analyzing a superposition of frequencies is called "spectral analysis" or "frequency domain analyses". Other instruments compare a superposition of signals from two different sources without any frequency domain conversion.
Mass spectrometry is a form of spectroscopy that measures changes in the mass-to-charge ratio of charged particles resulting from collisions with gas molecules. A mass spectrometer converts an analyte into ions and molecules by increasing the analyte's ionization energy (that is, raising the analyte's temperature) to high enough levels that thermal energy overcomes intermolecular bonds. Analyzers then isolate those ions according to their m/z values and measure their abundance. This enables an analyst to determine the chemical composition and relative abundance of each compound present in a sample, similar to chromatography.
The mass spectrometer must be calibrated by measuring the abundance of different molecules, which can be done with known standards. This calibration is done by measuring the abundance of the molecules present at different temperature values, using a mixture of ions and molecules. High temperatures are used to break up intermolecular bonds, then a lower temperature is used to measure the m/z of each component molecule at that temperature. This process is called "differential ionization". Replacement of ions by other groups (such as deuterium) also enables an analyst to measure the relative abundance of individual isotopes.
Matrix-assisted laser desorption/ionization (MALDI) is another form of spectroscopy that generates ions. However, the ions are not generated from a gas phase. Instead, the analyte of interest is first mixed with a chemical matrix to facilitate ionization. The matrix is usually an organic compound with a large amount of chromophore and a high thermal energy. This compound facilitates electron excitation which results in the dissociation of an analyte's chemical bonds and creation of ions or molecules. The spectrum and abundance measurement of these newly-created ions can be determined using the same methods used to analyze mass spectrometers.
In surface-enhanced Raman spectroscopy (SERS), a surface with a high number of adsorbed molecules is placed in close proximity to the sample being analyzed. The light source is then moved above the sample, which causes a shift in the light's frequency and intensity. This effect is caused by an interaction between photons of light and electrons on the analyte's surface. These vibrations within each molecule are subsequently transmitted to other molecules in the vicinity, resulting in a combined Raman signal from all of these molecules. This measured vibration is called a "Raman signal".
Time-resolved fluorescence involves analyzing fluorescence from an analyte over time. In Q-switching, a narrow band of energy (in the form of a laser pulse or series of pulses) is fired at a specific molecule, directing it to fluoresce. The fluorescence signal is then analyzed to identify the molecule and its abundance. A pulse can be divided into short segments, each of which is defined by its intensity and duration. As the number of segments increases, so does the resolution of measurements; however, this also reduces wavelength accuracy. Fluorescence lifetime analysis focuses on determining how quickly an analyte's intensity decays after the excitation pulse has ended. This measurement can be used in conjunction with other techniques to determine an analyte's molecular structure and how its environment affects its state.
Fluorescence lifetime imaging microscopy (FLIM) is a modern version of fluorescence lifetime analysis that uses an ultra-fast source to illuminate molecules within a sample. FLIM measures the frequency changes induced by the interaction of the excited states with molecules, which allows for better resolution of data. This technology can identify chemical differences within a sample on a microscopic level, which allows for more accurate data collection.
In general, fluorescent detectors are commonly used to measure many different light sources that can be quantified using fluorophores or fluorochromes. These light sources can range from ultraviolet, visible, and infrared light to X-rays, electrons and positrons. Fluorophores are chosen based on the type of radiation they absorb best, which is usually based on their electronic structure, especially the number of d-electrons and orbitals. They can be positively charged or neutral. Positively charged fluorophores are attracted to negatively charged surfaces when placed in a solution or mixed in a polymer using electrophoretic mobility.
Fluorochromes are molecules with two molecular groups of different colors that may react with other molecules. These molecules can be modified at their ends or mixed together to create a spectrum of colors, which is dependent on the energy levels within each molecule. Their most common color is red-green-blue, or RGB.
A fluorescent luminescent diode (FALD) is a device that emits light in different colors as a function of voltage, energy or current. Fluorescent diodes are commonly used in industry to produce light in specific wavelengths and intensities within a spectrum of colors used by other methods. The end product of FALDs are visible red, green and blue wavelengths that can be further processed using filters to produce other colors such as white, yellow and purple. This conversion allows FALDs to serve as more effective radiation sources than traditional broad-spectrum LEDs.
Fluorophores are used by FALDs as the basis of their emission spectrum, which is the intensity and wavelength of the light emitted by a device. One way to differentiate between fluorophores is their fluorescence endpoints and fluorescence lifetimes. Fluorescence lifetime can be described with reference to an exponential decay curve. This method allows for knowing how fast an analyte's fluorescence decays after being excited by a source. The decay curve can be represented as an open-topped S-shaped curve, or as a linear one for many different molecules. The fluorescence lifetime of a fluorophore is measured in femtoseconds (fs). This is the time it takes for a molecule's fluorescence to decay by a factor of ten. In addition, the luminescence lifetime is the main part of light emission that determines if a device can be distinguished from other devices.
A one-photon excitation fluorophore uses only one photon to excite an electron from its ground state to an excited state, which does not require energy absorption work. However, these molecules also have limitations in their fluorescence lifetimes. This type of fluorophore has excited states that are stable even after photon absorption has occurred. This arises from the fact that the molecule does not have enough energy to relax to its ground state, which requires work. These fluorophores are best for measuring low intensities of light, and are used in position-sensitive cameras.
Fluorophores have also been modified to produce higher fluorescence lifetimes using a process called fluoro-erosion. This method uses two or more reactive groups attached to a fluorophore in order to inhibit its fluorescence lifetime by dealing with the molecule's electronic structure. These molecules' lifetimes can be altered as long as they do not have an electron in their lowest energy state, which is different from fluorophores that require work to return an electron to its ground state.
In order for fluorophores to be used in sensors, it is necessary to measure the polarization state of the emitted light. Polarization states are useful in fluorescence because they can be used to identify different molecules that have the same emission spectrum. This is especially true in suspended particulate systems, which is why many sensors detect and differentiate between suspended particles using polarization sensitive measurements. A sensor's detector must be able to detect small differences in polarization states, which requires high levels of sensitivity within a single detector. In industrial and commercial applications, it is common for multiple detectors within a single system to output responses that are averaged by software after the detectors are read by a microprocessor or some other electronic controller.
The variation of fluorescence lifetimes from excited state to excited state can be used to detect the presence of a particle in a synthetic sample. This can be accomplished by selecting fluorophores that have different excitation energy levels, or are modified in such a way as to decay at different rates. These methods allow for greater separation between molecules that differ in fluorescent properties. Thus, it is possible to see a difference between “red” and “blue” particles within the same sample.
